Specific binding methods for the determination of enzymes have become firmly established. These methods are to be distinguished from methods where the catalytic activity of an enzyme is followed by measuring the action of the enzyme on its substrate, either by disappearance of the substrate or appearance of a product. Specific binding methods use no enzyme substrate, but instead employ a binding partner for the enzyme which is capable of reversibly, noncovalently binding the enzyme. Antibodies are oridinarily used as such binding partners because they are simple to make and handle. However, other binding partners such as cell surface receptors or carrier proteins may be useful.
Specific binding methods for the determination of enzymes may employ any of the routine procedures used previously for specific binding assays of non-enzyme antigens. For example, the well known sandwich or competitive immunoassays, as well as the homogeneous or heterogeneous enzyme-linked immunoassays, may be used. Such methods are routinely practiced in clinical laboratories using commercial kits containing all the reagents necessary to conduct the assay, e.g. labelled sample analogue, binding substance and standards. In the case of competitive immunoassays, however, it is preferred to insolubilize the antibody-bound enzyme after the sample and tracer enzyme have competed for limited antibody sites in solution. Insolubilization is conventionally accomplished by precipitating the immune complex of antibody and enzyme with a second antibody directed against the enzyme-binding antibody.
All of the labels heretofore used in specific binding assays may be employed for the determination of enzymes. Radioisotopes, stable free radicals, fluorescent and chemiluminescent compounds, enzymes, and coenzymes are all satisfactory, although enzyme and coenzyme labels should be selected to minimize interference from the enzyme of interest or enzymes in the sample to be determined.
Prostatic acid phosphatase (PAP) is now routinely determined by immunoassay. PAP (orthophosphoric monoester phosphohydrolase, acid optimum, E. C. 3.1.3.2) is a glycoprotein of approximately 100,000 daltons molecular weight. It is a major constituent of seminal fluid, normally secreted into semen and urine, and is present in serum at low levels. The acid phosphatases in general are distributed widely in the human body, and were first identified in human erythrocytes. They are present primarily in platelets, but also found in other blood cell components and maturing cells in the bone marrow as well as the epithelial cells and secretions of the prostate gland. The serum acid phosphatase level is normally a mixture of the enzymes contributed by the various tissue cells.
Discovery several decades ago that serum acid phosphatase activity is elevated in metastatic cancer of the prostate led to spectrophotometric assays for PAP activity which follow the enzymatic, or catalytic activity of PAP. Drawbacks to these assays are numerous. For example, lack of specificity for PAP is only partially overcome by the use of differential substrates and inhibitors. The major limitation is that these methods have generally been useful only for detection of metastatic prostatic cancer, and do not show adequate sensitivity for detection of the more curable earlier stages.
PAP immunoassays have been an improvement over the previous enzyme assays. They are more specific for the prostatic isoenzyme and exhibit greater sensitivity than the earlier enzyme assays, thus reportedly aiding in the more reliable detection of premetastatic prostatic cancer. Thus, excellent quantitation of serum PAP has been made practical by the use of immunoassay.
One advantage that has been urged in support of immunological rather than enzymatic methods for determining PAP is that the enzyme is immunologically stable, even though enzymatic acitivity may be rapidly lost. Thus no special steps have been used to preserve the PAP activity of samples as measured by enzyme assay. Foti et al., "Clinical Chemistry" 23 (1):9599 (1977), disclose that PAP is enzymatically unstable at 23.degree. C., for only about 25% of the starting PAP activity can be detected by an enzymatic assay, after sample storage for 48 hours at 23.degree. C. The enzyme instability notwithstanding, according to Foti et al. the conventional PAP determination by radioimmunoassay will produce constant PAP values.
Foti et al. do disclose some immunological activity losses at 37.degree. C. However, since assays generally wil be conducted at room temperature, i.e., close to 23.degree. C., Foti et al. concluded that "serum samples do not require special treatment (e.g., pH adjusting) and handling" before the PAP assay is conducted. Incidently, the pH adjustment mentioned by Foti et al. is a conventional step used in the earlier enzymatic assays for PAP: It has been considered good practice to acidify samples prior to PAP enzymatic assays because the enzyme activity is preserved at low pH.
Foti et al. also disclose the use of L-tartrate in enzymatic PAP assays. L-tartrate is a specific inhibitor for PAP which has the advantage that it does not appreciably inhibit other phosphatases. This phenomenon has been employed in PAP enzyme assays by first determining the total phosphatase activity of the sample and then the sample activity in the presence of L-tartrate. The difference between the two determinations is taken as the PAP activity. Foti et al. disclose that L-tartrate and other PAP inhibitors such as oxalate or fluoride do not interfere in the PAP immunoassay.
The conclusion of Foti et al. that PAP enzymatic activity is irrelevant to PAP immunoassay has been generally embraced by the art, but not without some confusion. For example, Endocrine Sciences' direction insert for its PAP radioimmunoassay unequivocally states that "detection of prostatic acid phosphatase by RIA is not dependent upon retention of enzyme activity in the serum sample", yet later observes that "PAP stability is decreased at room temperature . . . values were found to be somewhat lower after two days . . . samples for PAP analysis, therefore, should not be left at ambient temperatures for prolonged periods." Regardless of whether or not previous workers have recognized the immune instability of PAP, the concensus appears to be that enzyme stabilizers such as acid should not be added to the samples, standards or controls to be used in PAP immunoassays. Representative of this belief is Wang Laboratory's admonishment in its PAP radioimmunoassay direction insert that "(sample) sera should not contain any additive or acidifying agent." Similarly, while the New England Nuclear PAP RIA direction insert provides for reconstitution of lyophilized PAP tracer in pH 5.1 buffer (at which PAP is stable), it specifies a pH 7.4 buffer for reconstitution of the PAP standards.
Surprisingly, it has been found that the previously held beliefs of the prior art regarding PAP immunological stability have been incorrect. PAP is immunologically recognized by antibodies elucidated by the enzymatically active enzyme only for so long as it remains in the enzymatically active form. In fact, the decay of immunological activity closely parallels enzyme activity decay. The result of this misconception is that immunoassays currently being sold are capable of detecting only about 50% to 80% of the enzymatically inactive form of PAP. It is likely that serum samples will lose PAP enzyme activity at a different rate than PAP standards because of differences in collection and processing. Where such a discrepancy exists, the resulting assays will yield incorrect PAP values. For example, since standards heretofore generally have been supplied lyophilized and since the prior art instructions have been to include no additives or acid stabilizers in test samples, it is probable that the loss of PAP immunological activity in samples has exceeded that in standards. An assay conducted under these circumstances will yield artifically low PAP values. Even if by chance the standards and samples had suffered the identical degree of PAP inactivation at the time of assay, the resultant lower effective analyte concentration for both standards and sample would reduce the overall assay snesitivity. These unfortunate results neutralize the principal advantages espoused for PAP immunoassay: Reliable and sensitive PAP detection. The Endocrine Sciences' suggestion that PAP samples not be permitted to remain at ambient temperatures for prolonged periods, i.e., two days, is not adequate to fully protect the integrity of the assay because unacceptable losses in PAP immunological activity occur during this period.